Ormation of different chromatin structure after nucleosomes assembly. Nucleosomes assembly in
Ormation of different chromatin structure after nucleosomes assembly. Nucleosomes assembly in Stattic chemical information region 1, containing the 5S-G5E4 fragment, leads to dense, highly organized and stable chromatin. In contrast, assembly in region 2, corresponding to the pBSK-Zeo backbone, allows the formation of nucleosomes in a less dense, more dynamic and less organized chromatin structure. The overall chromatin structure of the plasmid was first checked by DNase 1 protection after nucleosomes assembly with increasing amounts of the native core histones, H3, H4, H2A and H2B (Additional file 2: Figure S2B). Then, typical restriction assays (REA) using restriction enzymes cutting distinctively in both regions (see Additional file 2: Figure S2A and S2C) showed that the 5S-G5E4 containing domain 1 was poorly accessible, unlike region 2 which remained highly sensitive to DNA cleavage. This confirmed the two distinct chromatin structural regions (regions 1, densely occupied and region 2 sparsely occupied), as predicted from the nucleosome positioning prediction algorithm. Finally, the accurate nucleosome positioning in the 5S-G5E4 region 1 was confirmed by agarose nucleosome gel shift assay performed on EcoRI cleaved 5S fragments (Additional file 2: Figure S2D), demonstrating the formation of evenly spaced mononucleosomes of the same size accurately PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28154141 positioned in known sites. In order to compare the different retroviral INs enzymes were all purified following similar purification procedure derived from previously published work [47] and tested under similar reaction conditions when possible. We first selected reaction conditions allowing efficient concerted integration for all the enzymes tested in the work. Two distinct assays have been reported by different groups to reproduce efficiently concerted integration in vitro. The first assay uses long viral DNA substrates and PEG and allows the detection of concerted integration in the absence of LEDGF/p75 but show poor stimulation effect of the cofactor [48,49]. The second assay developed by Cherepanov group does not use PEG and allows the detection of LEDGF/p75 dependent stimulation of concerted integration but poor PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28242652 full site integration in the absence of the cofactor [50,51]. Consequently, since our main aim was to analyze integration in the lack of cofactors, we have chosen to use long donor substrates in presence of PEG. These conditions allowed us to test HIV-1, PFV and MLV INs using the same final protein concentration of 100 nM. Blunt-end donor DNAs containing the specific viral endsBenleulmi et al. Retrovirology (2015) 12:Page 4 ofFigure 1 pBSK-Zeo-5S-G5E4 (p5S) acceptor plasmids used in the work. The 2.56 kb 5S-G5E4 fragment DNA for polynucleosome assembly (PN) was previously described [62] and was cloned into the pBSP-zeo vector (A). This fragment is made of two times five repeats of 5S sequences surrounding a central sequence containing five gal4 DNA binding sites and the adenovirus 2 E4 minimal promoter. The p5S vector thus contains a region 1 containing nucleosome-positioning sequences and a region 2 containing the pBSK-zeo backbone. Each 5S fragment is separated by two EcoRI restriction sites. Nucleosome occupancy prediction performed using the method previously described by [53] and used in [36] indicates the formation on the chromatinized vector of two regions with different chromatin organization (regular and stable nucleosomes in the 5S-G5E4 region 1 and less organized and stable nucleosomes in the.